The bacteriophage P22-based challenge phage system was used to study the binding of integration host factor (IHF) to its H' recognition site in the attP region of bacteriophage lambda. We constructed challenge phages that carried H' inserts in both orientations within the P22 Pa", promoter, which is required for antirepressor synthesis. We found that IHF repressed expression of Pa", from either challenge phage when expressed from an inducible P,,, promoter on a plasmid vector. Mutants containing changes in the H' inserts that decrease or eliminate IHF binding were isolated by selecting challenge phages that could synthesize antirepressor in the presence of IHF. Sequence analysis of 31 mutants showed that most changes were base pair substitutions within the H' insert. Approximately one-half of the mutants contained substitutions that changed base pairs that are part of the IMF consensus binding site; mutants were isolated that contained substitutions at six of the nine base pairs of the consensus site. Other mutants contained changes at base pairs between the two subdeterminants of the H' site, at positions that are not specffied in the consensus sequence, and in the dA+dT-rich region that flanks the consensus region of the site. Taken together, these results show that single-base-pair changes at positions outside of the proposed consensus bases can weaken or drastically disrupt I1F binding to the mutated site.The integration host factor (IHF) of Escherichia coli was originally discovered as a protein present in crude extracts that is required for integration of phage lambda DNA into the host chromosome (14, 15). More recently, it has been shown that IHF participates in a variety of other processes, including control of gene expression, plasmid replication, transposition, and the packaging of viral DNA into capsids (for a review, see reference 6).IHF is a sequence-specific DNA-binding protein, and a comparison of several IHF binding sites has led to the determination of a consensus recognition sequence; WATC AANNNNTTR (where W is A or T, N is any nucleotide, and R is A or G). In addition, IHF bends DNA upon binding (11,16,18,26,28,31,32); it is possible that its primary role in phage lambda recombination and other processes is to bend the DNA into a functional conformation (8,30).IHF is a heterodimer (Mr 20,000) composed of the gene products of the hip (himD) (Mr 9,000) and the himA (Mr 11,000) genes. Sequence analysis of the hip and himA (5, 21) genes showed that they are related to each other as well as to the HU protein and to transcription factor 1 of bacteriophage SPOl (for reviews, see references 4 and 10). The latter two proteins are basic and bind nonspecifically to DNA. The details of IHF binding to DNA remain to be elucidated, but recent footprinting studies indicate that IHF, unlike most sequence-specific binding proteins, interacts primarily with the minor groove of the DNA (3, 18, 33).Among the IHF binding sites, the three in the attP region of phage lambda (Hi, H2, and H') are the best characterized...